This project is oriented toward evaluating the mechanistic role for cellular membrane potential as a part of the driving force for intestinal Na ion - dependent transport systems. The evaluation depends on employing isolated intestinal epithelial cells as an experimental model which offers unusual experimental advantages relative to intact tissue models. These include: 1) selective control of mucosal and serosal sugar transport systems, 2) accurate definition of gradient forming capability for Na ion-dependent systems, 3) opportunity for steady-state kinetic analysis of transport fluxes, 4) limitation of tight-junctional and other non-cellular flux pathways, and 5) methods for independent control of cellular chemical potentials for Na ion and electrical potentials and hence opportunity for exploring the role for each in maintaining transport capability. The work encompasses effort aimed at development of methods for utilization of carbocyanine dyes and hydrophobic cations for noninvasive measurement of membrane potentials. Recent work has emphasized limitations in usual procedures for measuring Na ion:sugar coupling stoichiometries and provided information that the true coupling stoichiometry is 2Na ion per sugar molecule. Work in the coming year will focus on experiments which will help distinguish between various transport models involving 2Na ion ions per carrier cycle. Other work will be aimed at defining the extent to which a diffusional flux route for sugar limits the steady-state gradient forming capability of the Na ion-dependent carrier and the mechanistic basis for regulation of Na ion-dependent transport by exogenous ATP.